ABSTRACT Despite a large accumulation of potentially anti-tumoral immune cells, glioblastoma (GBM) sustains its growth and progression by establishing an immunosuppressive microenvironment. Major clinical efforts in multiple advanced cancers are aligned toward activating T cells via immune checkpoint inhibition. These strategies are currently being evaluated for GBM, the most common primary malignant brain cancer, based on success in other cancers. However, these approaches have not been uniformly successful, likely due to an abundance of immunosuppressive mechanisms that counteract T cell-activating therapies. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells that accumulate in tumors and are elevated in patients refractory to immune checkpoint inhibitors. Monocytic (M-) and granulocytic (G-) MDSC subsets have different mechanisms of immune suppression. We observed that these cells are present within the GBM microenvironment, with M- and G-MDSCs displaying differential tumor penetration, and are associated with a poor patient prognosis. MDSCs respond to signals generated by tumor cells, including the secretion of macrophage migration inhibitor factor (MIF), and both M- and G-MDSCs express MIF receptors, although in different patterns. These tumor cell-MDSC interactions result in potent immune suppression, and targeting MDSCs to alleviate this immune suppression confers a survival advantage in pre-clinical GBM models. Given the high number of immunosuppressive MDSCs present in this tumor, activating T cells alone may not be sufficient to attenuate tumor growth. However, there may be an opportunity to generate a durable immune response by concurrently activating T cells in combination with inhibiting MDSCs. The first translational goal of this project is to assess the individual function of M- and G-MDSCs on GBM growth and the specific signaling mechanisms they utilize, including the MIF axis. The second translational goal is to determine the consequence of targeting the MIF signaling axis to attenuate MDSC function in conjunction with T cell-activating strategies to enhance immune activation to reduce tumor growth. Based on our findings and new preliminary data, we hypothesize that MDSC subsets respond to MIF signaling differently, resulting in differential function during GBM growth. We also hypothesize that targeting MDSCs via MIF will reduce immune suppression and enhance the efficacy of immune activating strategies. Using a newly developed in vitro co- culture system in combination with MIF pathway knockout mice and blood-brain barrier-penetrating clinically relevant inhibitors and pre-clinical models, we will test this hypothesis through the following specific aims. Aim 1 will test the hypothesis that MDSC subtypes differentially regulate GBM growth via distinct MIF signaling responses and immunosuppressive capacities. Aim 2 will test the hypothesis that MIF receptor inhibition will attenuate MDSC function and can be combined with immune activating strategies to reduce GBM growth. The long-term goal of this project is to target the mechanisms employed by MDSCs to suppress the immune system in combination with T cell-activating strategies to generate a more complete immune response against GBM.